The baculovirus expression vector system (BEVS) is one of the most widely used gene expression systems for eukaryotic proteins because the proteins are properly processed with protein modifications such as phosphorylation, acetylation, and glycosylation (Becker et al. 1994; Grunewald et al. 1996; Ng et al. 1993; Ng et al. 1994; Zavodzky and Cseh 1996; Zhao and Sane 1993). See also, Lucow, V. A. and Summers, M. D. “High level expression of non-fused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors, Virology, 170:311-39 (1988); and U.S. Pat. No. 4,745,051. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the basis of the BEVS owing to its ability to express high levels of protein from its polyhedrin gene (polh) promoter. To express proteins, cultured insect cells are infected by recombinant AcMNPV, which carries the gene encoding the protein of interest.
Although the interest, need, and development of more insect cell lines has grown exponentially since the first insect cell lines were created by Gao (Gaw) and by Grace (Gaw et al. 1959; Grace 1962), currently there are only a few cell lines commercially available for use with the BEVS. These include “Sf21” derived from Spodoptera frugiperda (Vaughn et al. 1977) and BTI-Tn-5B1-4 (“Hi-5”) cells derived from Trichoplusia ni (Granados et al. 1994; McKenna et al. 1998), along with a clonal isolate of Sf21 called Sf9 cells (Smith et al. 1985). A genetically engineered Sf9 cell line called “Mimic” is also available from Invitrogen. Mimic cells are engineered to express five mammalian genes encoding glycosyltransferases enabling the cells to synthesize complex mono-sialylated N-glycans typical of mammalian but not most insect cells (Hollister et al. 2002).
Whereas many proteins are expressed efficiently using one of these currently available cell lines, many other proteins are expressed poorly, if at all. The reasons some proteins are poorly expressed in BEVS is not understood. In some cases, a change in the cell line used results in improved expression (Akermoun et al. 2005; Chai et al. 1996; Ogonah et al. 1996; Palomares et al. 2003). Thus a broader selection of cell lines available for BEVS is needed, and would be expected to increase the versatility of this expression system.
Membrane proteins are a major class of protein that is difficult to express to high levels in any heterologous expression system, but have been expressed with some success using BEVS (Akermoun et al. 2005; Grisshammer and Tate 1995; Massotte 2003; McCusker et al. 2007; Sarramegna et al. 2003). Sequencing the human genome revealed that membrane proteins make up approximately 30% of all of the encoded proteins, yet the structures for less than 3% have been solved. Membrane proteins are crucial for transport and signal transduction across membranes and play important roles in many medical conditions such as cancer, hypertension, and mental illness, thus they are major and important targets for drug development. Knowledge of membrane protein structure and function is critical for understanding their roles in these conditions and for drug development. Membrane proteins also are key targets for insecticides. Understanding how the structures of potential insecticide targets differ from their vertebrate counterparts also would be invaluable for developing new and safer chemicals for use in agriculture.
Most membrane proteins are not sufficiently abundant in nature to purify and crystallize and no gene expression system has been able to reliably express membrane proteins in sufficient quantities for these studies. The BEVS is efficient at expressing both cytoplasmic and secreted proteins and has been used to successfully express some membrane proteins at high levels (Grisshammer and Tate 1995; Massotte 2003). However, despite some success, many membrane proteins cannot be expressed at high enough levels for structural and functional studies.